PROJECT SUMMARY Our understanding of the properties of broadly-neutralizing antibodies (bnAbs) directed towards conserved influenza virus epitopes has rapidly evolved in the last five years and yet, we are still unraveling key features and functions of this antibody class. The overarching goal of this entire Program Project Grant is the generation of long-lived, broadly protective immune responses to influenza virus by vaccination. To this end, our group has developed novel immunogens and vaccination regimens that have yielded promising results in animal models and candidate vaccines based on chimeric hemagglutinins (cHA) are now advancing towards Phase I clinical trials. The work proposed in Project 1, seeks to advance the development of next-generation immunogens for group 2 influenza A viruses (IAVs) and influenza B viruses (IBVs), in an effort to overcome the immunodominance of the globular head domain of HA, and allow improved recognition of highly conserved, but immunosubdominant HA epitopes. The application of our prior expertise with influenza A cHA immunogens will allow us to rapidly advance new group 2 cHA constructs. However, the development of a truly universal influenza virus vaccine will require combined efforts to elicit bnAb responses against group 1 and 2 IAVs as well as IBVs. Therefore, we propose to generate new universal influenza B virus constructs, based on novel mosaic HA design in which major antigenic sites in the immunodominant HA head domain have been eliminated. Optimized cHA for group 2 IAVs, or mosaic immunogens for IBVs, will be tested as DNA, recombinant protein, inactivated influenza vaccine (IIV) or live attenuated influenza vaccine (LAIV) formulations in mice for their ability to elicit broad and protective humoral immune responses recognizing conserved epitopes. In parallel, we will continue to further elucidate the determinants of Fc-mediated protection using novel techniques to probe the interactions between broadly protective HA antibodies, infected target cells, and effector cells. These mechanisms will be extended to investigations of the role of antibodies recognizing additional influenza viral antigens, including the viral neuraminidase (NA), in collaboration with Project 4. This program of work will be executed through close collaborations with each of the other Projects. We will characterize human and murine monoclonal antibodies (mAb) produced by Project 4 and the mAb Core. Vaccine constructs will be formulated with adjuvants selected in Project 2 to see if we can enhance Ab responses directed towards conserved epitopes and the in vivo efficacy of lead vaccine immunogens will be evaluated in the ferret model in Project 3. Thus, the overall aim of our project is to identify unique properties of broadly protective immune responses that can be exploited to further enhance and aid in the development of universal influenza virus vaccines.